clinical and biological implications of cd133-positive and cd133-negative cells in glioblastomas

Clinical and biological implications of CD133-positiveand CD133-negative cells in glioblastomasKyeung Min Joo1,2,7, Shi Yean Kim1,2,7, Xun Jin3, Sang Yong Song4, Doo-Sik Kong1, Jung-II Lee1, Ji Won Jeon1,2,Mi Hyun Kim1,2, Bong Gu Kang1,2, Yong Jung1,2, Juyoun Jin1,2, Seung-Chyul Hong1, Woong-Yang Park5,Dong-Sup Lee6, Hyunggee Kim3 and Do-Hyun Nam1,2

A number of recent reports have demonstrated that only CD133-positive cancer cells of glioblastoma multiforme (GBM)have tumor-initiating potential. These findings raise an attractive hypothesis that GBMs can be cured by eradicatingCD133-positive cancer stem cells (CSCs), which are a small portion of GBM cells. However, as GBMs are known to possessvarious genetic alterations, GBMs might harbor heterogeneous CSCs with different genetic alterations. Here, we com-pared the clinical characteristics of two GBM patient groups divided according to CD133-positive cell ratios. The CD133-low GBMs showed more invasive growth and gene expression profiles characteristic of mesenchymal or proliferativesubtypes, whereas the CD133-high GBMs showed features of cortical and well-demarcated tumors and gene expressionstypical of proneuronal subtype. Both CD133-positive and CD133-negative cells purified from four out of six GBM patientsproduced typical GBM tumor masses in NOD-SCID brains, whereas brain mass from CD133-negative cells showed moreproliferative and angiogenic features compared to that from CD133-positive cells. Our results suggest, in contrast toprevious reports that only CD133-positive cells of GBMs can initiate tumor formation in vivo CD133-negative cellsalso possess tumor-initiating potential, which is indicative of complexity in the identification of cancer cells fortherapeutic targeting.Laboratory Investigation (2008) 88, 808–815; doi:10.1038/labinvest.2008.57; published online 16 June 2008

KEYWORDS: cancer stem cell; CD133; clinical manifestation; gene expression profile; glioblastoma

A recent concept in brain tumor biology is that brain tumorsarise from cancer stem cells (CSCs) that are CD133 positive(CD133(þ )). It has been reported that a small number ofCD133(þ ) glioblastoma multiforme (GBM) cells are able torecapitulate the original tumor in vivo, whereas millions ofCD133-negative (CD133(�)) cells could not produce braintumor masses.1–6 However, accumulating evidence suggests thatCD133(�) GBM cells can also regenerate heterogenous tumorsin vivo,7,8 and generation of the huge and rapidly growingtumors by only CD133(þ ) CSCs would be difficult becausemore than 50% of GBM patients have few CD133(þ ) cells.9

As a majority of neurogenic astrocytes in the adult brainare not recognized by a CD133 antibody,8 it is likely that

CD133 might be newly expressed in GBM CSCs that arederived from CD133(�) adult neural stem cells (NSCs) orterminally differentiated brain cells, such as astrocytes, neu-rons, and oligodendrocytes. Given that the gene expressionprofile is changed when GBM recurs after treatments,10 it isplausible that new CD133 expression may occur if thecharacteristics of CSCs are changed or if some CSCs are se-lected by treatment. Furthermore, the wide-range variationin CD133(þ ) cell ratio (0.1–50% in GBM patients)1–6

also suggests the existence of other GBM CSCs that do notexpress CD133.

Therefore, we hypothesize that there are several kinds ofCSCs in the tumor mass of GMB, and these diverse CSCs

Received 09 March 2008; revised 03 April 2008; accepted 03 April 2008

1Department of Neurosurgery, Samsung Medical Center and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, South Korea,2Cancer Stem Cell Research Center, Samsung Medical Center and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, SouthKorea; 3School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea; 4Department of Pathology, Samsung Medical Center and Samsung BiomedicalResearch Institute, Sungkyunkwan University School of Medicine, Seoul, South Korea; 5Department of Biochemistry and Molecular Biology, Seoul National UniversityCollege of Medicine, Seoul, South Korea and 6Department of Anatomy, Seoul National University College of Medicine, Seoul, South KoreaCorrespondence: Dr Do-H Nam, MD, PhD, Department of Neurosurgery, Samsung Medical Center and Biomedical Research Institute, Sungkyunkwan University Schoolof Medicine #50, Irwon-dong, Gangnam-gu, Seoul 135-710, South Korea or Dr H Kim, PhD, Cell Growth Regulation Laboratory, School of Life Sciences andBiotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, South Korea.E-mail: [email protected] or [email protected]

7These authors contributed equally to this work.

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possess diverse cellular and molecular characteristics, such astumor initiation potential, chemo-radiation resistance, andgene expression profile.

MATERIALS AND METHODSTumor Specimens and Primary Tumor CulturesFollowing informed consent, tumor samples classified asGBMs based on World Health Organization criteria11 wereobtained from patients undergoing surgical treatment at theSamsung Medical Center (Seoul, Korea) in accordance withthe appropriate Institutional Review Boards. Tumors wereenzymatically dissociated into single cells and red blood cellswere removed by differential centrifugation. Dissociated cellswere cultured in the ‘NBE’ conditions consisting of Neuro-Basal Media (Invitrogen, Carlsbad, CA, USA), N2 and B27supplements (Invitrogen), as well as human recombinantbFGF and EGF (50 ng/ml each; R&D Systems, Minneapolis,MN, USA). To differentiate cultured cells, cells were platedonto poly-L-lysine/laminin mixture-precoated culture dish(Invitrogen) and were subjected to growth in DMEM with10% fetal bovine serum (10% FBS/DMEM; Cambrex, EastRutherford, NJ, USA). Immunofluorescence assay wasperformed using following antibodies: Tuj1 (Chemicon,Billerica, MA, USA), GFAP (Sigma, St Louis, MN, USA), andO4 (Chemicon).

Fluorescence-Activated Cell Sorting and Intracranial CellTransplantationDissociated GBM cells were labeled with 10 ml anti-CD133/2-PE antibody (Miltenyi Biotec, Bergisch Gladbach, Germany)per 106 cells. CD133(þ ) cell ratios were analyzed, andCD133(þ ) and CD133(�) populations were sorted using aFACSAria machine (BD Biosciences, San Jose, CA, USA). Atotal of 5� 103–2� 104 purified cells suspended in 5 ml HBSSwere stereotactically injected into 6-week-old NOD/SCIDmouse brains (2 mm left and 1 mm anterior to the bregma,2 mm deep).

Specimens and ImmunohistochemistryAs shown neurological symptom, mice were sacrificed andtheir brains were removed. The brains were processedfor paraffin or frozen section. Paraffin sections were stainedwith hematoxylin and eosin as per standard histopatho-logical technique and the tumor volume was recorded (lar-gest width2� largest length� 0.5). Frozen sections were fixedwith acetone and then immunohistochemistry was per-formed as described previously12 using following antibodies:CD31 (BD Pharmingen, San Diego, CA, USA), PCNA (Dako,Glostrup, Denmark), and p-Akt (Cell signaling technology,Danvers, MA, USA).

RNA Expression Array and Data AnalysisTotal RNAs were isolated using TRIZOL reagent (Invitrogen)and further purified using RNeasy Mini Kit (Qiagen,Hilden, Germany). Samples were processed and hybridized to

Affymetrix U-133 plus2 GeneChip Arrays according to theAffymetrix GeneChip Expression Analysis Technical Manual(Affymetrix, Santa Clara, CA, USA). All arrays were confirmedto be within acceptable minimal quality control parameters.The gene expression CEL files were normalized using RobustMultichip Averaging procedure and PM-MM difference modelwas used to obtain the expression values. The hierarchicalcluster analysis was performed using R package software 2.6.0using the Euclidean distance with complete linkage.

RESULTSGBM Patients Divided into CD133-High and CD133-LowGBM Groups Show Different Clinical CharacteristicsTo evaluate our hypothesis, we first divided GBM patientsinto a CD133-high group (CD133(þ ) cell ratio Z3%, n¼ 7)and a CD133-low group (CD133(þ ) cell ratio o3%, n¼ 13)by fluorescence-activated cell sorting (FACS) analysis withanti-CD133 antibody (Supplementary Figure 1, Table 1). Weanalyzed the patients’ clinical characteristics using MRI scan data(Figure 1a) to determine whether these groups possess similar ordifferent clinical characteristics. As compared to CD133-highGBMs, CD133-low GBMs have tendency to be localized withinthe deeper structures of the brain and to show more invasivegrowth patterns and ventricle involvement (Figure 1b). However,there was no significant difference in sphere-forming capacitybetween cells derived from CD133-high and CD133-lowGBMs, as cultured under ‘NBE’ conditions (NeuroBasal Mediasupplemented with N2, B27, EGF, and bFGF) (Table 1).

CD133-Low and CD133-High GBMs Show DifferentClinical OutcomesAs CD133-low GBMs showed more invasive morphologies inMRI scan, we tested whether disease progression after treat-ment is different between CD133-high and CD133-low GBMpatients. The rate of disease progression after chemotherapyand radiotherapy was relatively higher in the CD133-lowGBMs (45.5%) compared to CD133-high GBMs (28.6%)(Figure 1c). Therefore, these results indicate that CD133-highand CD133-low GBM patients have different clinical char-acteristics and outcomes.

CD133-Low and CD133-High GBMs Show Different GeneExpression ProfilesNext, we compared gene expression patterns between sub-groups of patients whose GBMs had the lowest and highestCD133(þ ) cell ratios, termed ‘CD133-lowest’ (CD133(þ ) cellratio o0.5%, n¼ 3) and ‘CD133-highest’ (CD133(þ ) cellratio 415%, n¼ 4). Following cDNA microarray analysis, weperformed a hierarchical clustering of 34 selected genes thatare used for subclassification of GBM, such as proneuronal,mesenchymal, and proliferative subtypes.10,11 As shown inFigure 2a, CD133-lowest GBMs showed a relative upre-gulation of genes that have been previously linked to the‘mesenchymal’ or ‘proliferative’ subtype, which has worseclinical outcome, whereas CD133-highest GBMs showed

CD133 in glioblastoma

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higher expression of genes that are known to be upregulatedin the ‘proneuronal’ GBM subtype.10 These results werefurther validated by real-time RT-PCR, showing that ex-pression of DLL3 and SOX8 (proneuronal genes) is de-creased, while YKL40 (a mesenchymal gene) was upregulatedin the CD133-lowest GBMs (Figure 2b).

Previous data showed that when dissociated GBM cells arecultured in ‘NBE’ conditions, which were originally designedto culture normal NSCs, they become very similar to normalNSCs and thereby recapitulate the genotype, gene expressionpatterns, and in vivo biology of GBMs.13 Interestingly, whencells dissociated from CD133-high GBM patients (no. 16 or19) (CD133(þ ) cell ratios¼ 19.90 and 45.54%, respectively)were subjected to growth in ‘NBE’ conditions for 10 days,expression of genes characteristic of the ‘proneuronal’ GBMsubtype was markedly decreased, whereas expression of‘mesenchymal’ or ‘proliferative’ GBM subtype-related genesincreased (Figure 2c). However, both CD133(þ ) andCD133(�) GBM cells grown in 10% FBS/DMEM conditions

for 10 days were prone to differentiate into various brain cellsthat express GFAP (astrocyte), Tuj1 (neuron), or O4 (oligo-dendrocyte) (data not shown). These results indicate that thegene expression pattern of CSCs can change depending onin vivo and in vitro conditions, without alteration of theirdifferentiation property.

Both CD133-Low and CD133-High GBMs Show StemCell-Like Characteristics In VitroWe compared the sphere-forming abilities of the CD133-lowor CD133-high GBMs in the ‘NBE’ condition and found that9 in 13 CD133-low GBMs (69%) made many spheres withina week (Figure 3a, Table 1). This frequency was comparablewith that of CD133-high GBMs (six in seven, 86%). Even thethree CD133-lowest GBMs (patient nos. 1, 2, 3; CD133(þ )

cell ratio o0.2%) made spheres well in vitro (Table 1).Spheres formed by either CD133-low or CD133-high GBMshad similar morphology and size (Figure 3a). When thesphere cells were forced to differentiate in 10% FBS/DMEM,

Table 1 Summary of patients and their clinical characteristics

Patientno.

Sex Age (years) Pathologicalsubtype

% CD133(+)

tumor cellsaTumorlocationb

Necrosisb Mult. orinvas.b

Ventricleinvol.b

Leptomenin.spreadingb

Gliomabackc

Primary orsecondary

Sphereformationd

1 F 59 Glioblastoma 0.01% Deep Y Y Y N N Primary B

2 M 60 Glioblastoma 0.05% Cortex Y N N N N Primary B

3 M 56 Glioblastoma 0.12% Deep Y Y Y N Y Secondary B

4 F 40 Glioblastoma 0.19% Deep Y N Y N N Primary C

5 M 36 Glioblastoma 0.25% Deep Y N N N Y Secondary B

6 F 31 Glioblastoma 0.31% Deep Y N N N N Primary B

7 M 42 Glioblastoma 0.57% Cortex Y Y N N Y Primary B

8 M 33 Glioblastoma 1.22% Deep Y Y Y Y N Primary D

9 F 55 Glioblastoma 1.42% Deep Y Y Y N N Primary D

10 M 49 Glioblastoma 1.82% Cortex Y N N N Y Primary D


12 M 48 Glioblastoma 2.57% Deep Y Y Y N N Primary A


14 M 37 Glioblastoma 3.15% Cortex Y N Y N N Primary B

15 F 82 Glioblastoma 4.91% Cortex Y N N N N Primary B

16 M 57 Glioblastoma 19.90% Cortex Y N N N N Primary A

17 F 59 Glioblastoma 35.31% Cortex Y N N N N Primary B

18 M 65 Glioblastoma 41.70% Cortex N N N N N Primary B

19 F 54 Glioblastoma 45.54% Cortex Y N N N N Primary A

20 F 26 Glioblastoma 46.84% Cortex Y N Y N Y Primary C

Cortex, cerebral cortex; deep, deep seated; glioma back., glioma background; invas., invasiveness; leptomenin. spreading, leptomeningeal spreading;mult., multiplicity of tumor mass; ventiricle invol., ventricle involvement.a

Flow cytometry was used to determine the proportion of CD133(+) cells from each patient’s tumor mass.b

MRI scan data were used.c

Pathologically diagnosed.d

After 1� 106 acutely dissociated cells were cultured under sphere-forming conditions for 14 days, the number of spheres was analyzed. A: 450; B: 11–50;C: 1–10; D: 0.


KM Joo et al



Figure 1 CD133-high and CD133-low GBM patient groups show different clinical characteristics. (a) Representative MRI scan data of four CD133-low

patients (patient nos. 1, 3, 4, 5) and four CD133-high patients (patient nos. 16, 17, 18, 20) divided by CD133-positive cell ratio. (b) Comparisons

of tumor location, invasiveness or multiplicity, and ventricle involvement between CD133-high patients and CD133-low patients. *Po0.05.

(c) Comparison of disease progression rates between 11 CD133-low and seven CD133-high GBM patients after chemotherapy and radiotherapy.

Figure 2 CD133-lowest and CD133-highest GBMs show different gene expression profiles. (a) Heatmaps showing expression profiles of the genes

classified into ‘proneuronal’, ‘mesenchymal’, and ‘proliferative’ subtypes in three CD133-lowest and four CD133-highest GBM tumor masses.

(b) The expression levels of five selected genes (CD133, DLL3, SOX8, YKL40, and E2F7) in three CD133-lowest and four CD133-highest GBM tumor

masses were validated by real-time RT-PCR. *Po0.05. (c) Heatmaps showing the expression profiles of the genes classified into ‘proneuronal’,

‘mesenchymal’, and ‘proliferative’ subtypes in two CD133-high GBM tumor masses (patient nos. 16 and 19) and their cognate cells, which are

cultured in ‘NBE’ conditions for 10 days after acute dissociation from tumor masses.


KM Joo et al



most cells from both CD133-low and CD133-high GBMsshowed morphology of differentiated cells and expresseddifferentiated neural cell markers such as Tuj1, GFAP, and O4(Figure 3b; a representative data from GBM patient no. 12).These results suggest that cells from CD133-low GBMs alsohave stem cell-like characteristics in vitro.

Both CD133(�) and CD133(þ ) GBM Cells PossessTumor-Initiating PotentialAs it has been recently documented that some GBM patientshave CD133(�) CSCs and others have CD133(þ ),7 and low-grade gliomas possess multiple and spatially distinct clonal

populations,14 we isolated CD133(þ ) and CD133(�) cellsfrom tumor specimens of a number of GBM patients usingFACS analysis (Figure 4a; a representative data from GBMpatient no. 4) to compare tumor-initiating potential. WhenCD133(þ ) or CD133(�) cells purified by FACS sorting wereinjected into the brains of NOD/SCID mice (n¼ 3–5),tumors were produced in about half of the injected brains,indicating that both cell types harbor tumor-initiatingpotential (CD133(þ )¼ 7 in 17, CD133(�)¼ 11 in 20). Inaddition, both CD133(þ ) and CD133(�) cells purified fromfour out of six human GBM patients were able to generatetumor masses that show similar GBM histological features

Figure 3 Both CD133-low and CD133-high GBMs show stem cell-like characteristics in vitro. (a) Neurosphere-forming ability in the ‘NBE’ condition

was compared between cells dissociated from CD133-high and CD133-low GBMs. The representative photos of spheres derived from CD133-high and

CD133-low GBMs (left panel). (b) Sphere cells cultured in the ‘NBE’ condition were differentiated with 10% FBS. Representative photos present expressions

of Tuj1, GFAP, and O4 determined by immunofluorescence assay.


KM Joo et al



Figure 4 Both CD133(�) and CD133(þ ) GBM cells possess tumor-initiating potential. (a) Representative FACS data showing CD133(þ ) and CD133(�) cell populations isolated from one of 20 GBM

patients using FACS. (b) When orthotopically injected into NOD/SCID mice, both CD133(þ ) and CD133(�) cells derived from one GBM patient produced tumor masses. Representative photos

showing brain tumor tissue sections stained with hematoxylin and eosin. (c) Representative photos showing CD31-positive, PCNA-positive, and pAkt-positive cells in the tumor masses derived

from CD133(�) and CD133(þ ) cells. Quantitative data showing microvessel numbers, PCNA(þ ) and pAkt(þ ) cell numbers in tumors derived from CD133(�) and CD133(þ ) cells. *Po0.05.

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(Table 2, Figure 4b). Furthermore, tumor volumes in themice injected with sorted CD133(þ ) or CD133(�) cells werenot significantly different, and neurological symptomswere detected at almost the same time point from injections(Table 2). However, although the volume and overallhistology of tumor masses produced by injection of sortedCD133(þ ) or CD133(�) cells were relatively similar, thetumor masses from CD133(�) cells contained moremicrovessels and PCNA(þ )-proliferating cells (Figure 4c),whereas those from CD133(þ ) cells were found to havesignificantly higher levels of phosphorylated Akt (pAkt,Figure 4c). These results suggest that there are multiple kindsof tumor-initiating cells, such as CD133(�) and CD133(þ )

cells, in every GBM patient.

DISCUSSIONA new paradigm in brain tumor biology that only CD133(þ )

cancer cells harbor a tumor-initiating potential raises an at-tractive hypothesis that GBMs can be cured if one eradicatesCD133(þ ) CSCs, which occupy small portion of GMB cells.3,4 However, the story would not be simple, as GBMs havebeen known to possess various genetic alterations and toconsist of heterogeneous cell populations,15,16 arguing thatCSCs in GBMs might also be heterogeneous and possessdifferent genetic alterations. In support of above postulation,it has been recently documented that some GBM patientshave CD133(�) CSCs and others have CD133(þ ).7 Even low-grade gliomas, which show more relatively homogeneoushistology than GBMs, have been reported to possess multipleand spatially distinct clonal populations.14 Furthermore, inthe present study, we demonstrated that there are CSCs inboth CD133(þ ) and CD133(�) cell population originatedfrom one GBM patient, and both CD133(�) and CD133(þ )

cells enabled the formation of tumor masses that showedsimilar volumes at 3 months post-injections.

Consistent with a previous report that GBM patients canbe divided into groups that present different prognosis,10 wefound that GBM patients can be classified into two groups(CD133-low and CD133-high group) according to theirCD133(þ ) cell ratios, and CD133-low GBMs (CD133(þ ) cellratio o3%) showed more aggressive morphologies as de-termined by MRI scan and unique gene expression patterns(‘mesenchymal’ or ‘proliferative’ subtype) that are related toworse prognosis.10,11 It is interesting to note that it has beenreported that gene expression profiles change from the ‘pro-neuronal’ to ‘mesenchymal’ subtype in GBMs recurred aftertreatment.10 As CD133(�) CSCs represented the ‘mesenchy-mal’ type, it might be plausible that CD133(�) CSCs might bemore resistant to radiotherapy and chemotherapy comparedto CD133(þ ) CSCs, which are already recognized to bemarkedly resistant to conventional anticancer therapies.17,18

When GBM cells dissociated from GBM patients arecultured in the ‘NBE’ condition, which is originally designedto culture the normal NSCs, they harbor extensive similaritiesto normal NSCs and thereby recapitulate the genotype, geneexpression patterns, and in vivo biology of GBMs.13 As GBMcells dissociated from CD133-highest GBMs (CD133(þ ) cellratios¼ 19.90 or 45.54%), which presented ‘proneuronal’subtype of gene expression, were cultured in the ‘NBE’condition for 10 days, their gene expression patterns werechanged from ‘proneuronal’ subtype to ‘mesenchymal’ or‘proliferative’ subtypes that were observed from CD133(�)

CSCs, arguing that CD133(�) CSCs might have advantagesfor survival or proliferation in the ‘NBE’ condition.

Taken together, our data show that there are at least twokinds of CSCs (CD133(þ ) and CD133(�)) in each GBM

Table 2 Summary of tumor incidence, tumor volume, and the onset of first neurological symptom of mice injected with CD133(+)

and CD133(�) cells isolated from six GBM patients

Patientno.

Sortingmethod

Injected cellnumber

Tumorincidence

Tumor volume(mm3)

First neurologicalsymptom (days)

1 FACS CD133(+)¼ 1� 104 CD133(+)¼ 1/4 CD133(+)¼ 2.2 CD133(+)¼ 192

CD133(�)¼ 1� 104 CD133(�)¼ 1/3 CD133(�)¼ 6.6 CD133(�)¼ 239

3 FACS CD133(+)¼ 5� 103 CD133(+)¼ 0/3 — —

CD133(�)¼ 5� 103 CD133(�)¼ 0/3

4 FACS CD133(+)¼ 1� 103 CD133(+)¼ 1/2 CD133(+)¼ 42.8 CD133(+)¼ 139

CD133(�)¼ 2� 104 CD133(�)¼ 4/5 CD133(�)¼ 35.7±9.9 CD133(�)¼ 123.7±13.3

7 FACS CD133(+)¼ 5� 103 CD133(+)¼ 0/3 — —

CD133(�)¼ 2� 104 CD133(�)¼ 0/3

13 FACS CD133(+)¼ 5� 103 CD133(+)¼ 3/3 CD133(+)¼ 83.1±24.1 CD133(+)¼ 94.7±4.0

CD133(�)¼ 5� 103 CD133(�)¼ 3/3 CD133(�)¼ 73.7±6.7 CD133(�)¼ 97.3±5.8

18 FACS CD133(+)¼ 2� 104 CD133(+)¼ 2/2 CD133(+)¼ 113.3±18.4 CD133(+)¼ 90.5±5.0

CD133(�)¼ 2� 104 CD133(�)¼ 3/3 CD133(�)¼ 80.7±16.0 CD133(�)¼ 120.7±23.0

FACS, fluorescence-activated cell sorting; GBM, glioblastoma multiforme.


KM Joo et al



patient and that they have different biological andclinical characteristics. An understanding of all GBM CSCtypes and their clinical implications, as well as developmentof new GBM CSC-specific markers, should be a crucial steptoward more effective therapeutic modalities against humanGBMs.

Supplementary Information accompanies the paper on the Laboratory

Investigation website (http://www.laboratoryinvestigation.org)

ACKNOWLEDGEMENTS

This work was supported by the Korea Science and Engineering Foundation

(KOSEF) grant funded by the Korea government (MOST) (no.

MM10641000104-06N4100-1040 to DH Nam) and a grant from the National

R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of

Korea (0720030 to H Kim).

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clinical and biological implications of cd133-positive and cd133-negative cells in glioblastomas

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